Structure and activity of a class II lanthipeptide from a thermophilic bacterium

This study identifies and characterizes thermolanthin, a novel class II lanthipeptide from the thermophilic bacterium *Thermoactinomyces* sp. DSM 45891, which features unique DL-(methyl)lanthionine ring structures deviating from typical stereoselectivity and exhibits antimicrobial activity against Gram-negative ESKAPE pathogens.

The Gist

Imagine a microscopic factory inside a heat-loving bacterium (a "thermophile") that lives in scorching hot environments. This factory has a special assembly line designed to build tiny, super-strong molecular "handcuffs" that can lock onto and disable harmful bacteria.

This paper is the story of scientists discovering, reverse-engineering, and analyzing two of these molecular handcuffs, which they named Thermolanthin.

Here is the breakdown of the story using simple analogies:

1. The Blueprint and the Factory

The scientists found a set of instructions (a gene cluster) in a heat-loving bacterium called Thermoactinomyces. This blueprint told the cell how to make two very similar raw materials (precursor peptides), which they called TlaA1 and TlaA2.

Think of these raw materials as long, floppy strings of beads. On their own, these strings are useless. They need a master craftsman to transform them into something useful. That craftsman is an enzyme called TlaM.

2. The Master Craftsman (TlaM)

The enzyme TlaM is like a highly skilled tailor with a magical sewing machine. Its job is to take the floppy strings and stitch them together into tight, complex knots.

• The Process: First, the tailor dries out certain parts of the string (removing water molecules). Then, it uses a special needle to stitch the string back onto itself, creating strong, unbreakable loops called thioether rings.
• The Result: Instead of a floppy string, you get a rigid, 3D structure that looks like a molecular cage or a complex knot.

3. The Surprise Twist: Breaking the Rules

Usually, when these "tailors" stitch these knots, they follow a strict rule: they always stitch in a specific direction (like a right-handed screw). This is called "stereoselectivity."

However, the scientists discovered that TlaM broke the rules.

• The Analogy: Imagine a factory that only makes right-handed gloves. Suddenly, they start making left-handed gloves with the exact same machine.
• The Discovery: TlaM stitched the knots in a "left-handed" way (DL-stereochemistry) instead of the usual "right-handed" way. This was a huge surprise because the blueprint (the DNA sequence) looked like it should have produced the standard right-handed version. The enzyme is so powerful it forces the material to bend in a direction nature usually doesn't allow.

4. Two Peas in a Pod

The bacterium makes two very similar products (TlaA1 and TlaA2). They are like twins; they share about 58% of their DNA code.

• TlaA1 gets stitched into 4 tight loops.
• TlaA2 also gets stitched into 4 tight loops, but with a slightly different pattern near the end.
• The Mystery: Usually, when bacteria make two similar products, they need both of them working together to kill bad bacteria (like a two-person team). But in this case, the scientists found that TlaA1 could work all by itself once it was cut to the right size. It didn't need its twin to be effective.

5. The Weapon: Thermolanthin

The scientists took the modified TlaA1, cut off the "tag" that the cell uses to hold it, and tested it.

• The Result: This new molecule, which they named Thermolanthin, acted like a biological weapon. It successfully killed not only common bacteria but also some of the most dangerous, drug-resistant "superbugs" (known as ESKAPE pathogens) that usually cannot be stopped by standard antibiotics.
• Why it matters: Most of these molecular handcuffs only work on Gram-positive bacteria (like Staph). It is very rare for them to work on Gram-negative bacteria (like E. coli or Klebsiella), which have a tough outer armor. Thermolanthin managed to punch through that armor.

6. The "Hot" Advantage

Why study a bacterium that lives in heat?

• The Analogy: Think of enzymes from normal bacteria as plastic toys that melt if you leave them in the sun. Enzymes from heat-loving bacteria are like metal tools; they are built to withstand extreme temperatures and harsh conditions.
• The Benefit: Because these tools are so sturdy, they are easier to manufacture in a lab and might be more stable when used as medicines in the human body.

Summary

In short, this paper is about finding a new, super-strong molecular knot made by a heat-loving bacterium. The scientists figured out exactly how the knot is tied, discovered that the "tailor" enzyme breaks the usual rules of chemistry to make it, and found that the final product is a potent weapon against dangerous, drug-resistant superbugs. It opens the door to designing new antibiotics that are tough, stable, and effective against the world's most stubborn infections.

Technical Summary

1. Problem Statement

Lanthipeptides are a diverse class of ribosomally synthesized and post-translationally modified peptides (RiPPs) known for their antimicrobial properties (lantibiotics). While genome mining has identified numerous biosynthetic gene clusters (BGCs), many remain uncharacterized. Specifically:

• Two-component systems: Many lanthipeptide BGCs encode two precursor peptides. In known systems (e.g., lacticin 3147, haloduracin), these often act synergistically, with individual peptides lacking activity. However, the structural rules governing these systems are not fully understood.
• Thermophilic sources: Enzymes from thermophiles offer potential for enhanced stability and robustness, yet few lanthipeptides from thermophilic bacteria have been characterized.
• Stereochemical rules: Class II lanthipeptide synthetases typically follow a stereoselective rule where a specific motif (Dhx-Dhx-Xxx-Xxx-Cys, where Dhx is dehydroalanine or dehydrobutyrine) yields LL-(methyl)lanthionine rings. Deviations from this rule are rare and mechanistically unexplained.
• Knowledge Gap: The study aimed to characterize the tla BGC from Thermoactinomyces sp. DSM 45891, which encodes two highly similar precursor peptides (TlaA1 and TlaA2) and a single synthetase (TlaM), to determine if they form a two-component system, their unique ring structures, and their stereochemistry.

2. Methodology

The researchers employed a combination of bioinformatics, heterologous expression, mass spectrometry, NMR spectroscopy, and chemical derivatization assays.

• Bioinformatics: The tla BGC was identified via sequence similarity networks. It encodes two precursor peptides (tlaA1, tlaA2) with ~58% identity, a class II synthetase (tlaM), and a PCAT transporter (thtT).
• Heterologous Expression: The genes were co-expressed in E. coli. Precursor peptides were fused to His6-SUMO tags for purification. The synthetase TlaM was co-expressed to modify the precursors.
• Leader Peptide Removal: The modified precursors were cleaved in vitro using the C39 protease LahT150 (an ortholog of the native TlaT) to remove leader peptides and generate mature core peptides (mTlaA1 and mTlaA2).
• Chemical Assays:
  • NEM (N-ethylmaleimide) Assay: To determine if free cysteines remained (indicating incomplete cyclization).
  • DTT (Dithiothreitol) Assay: To detect unmodified dehydroamino acids (Dha/Dhb) by ring opening.
• Proteolytic Digestion & MS: Fragments were generated using chymotrypsin, GluC, LysC, and AspN. Tandem MS (MS/MS) was used to map dehydration sites and ring connectivity.
• NMR Spectroscopy: To resolve overlapping ring patterns that MS could not distinguish, specific fragments (Fragment 5 from mTlaA2 and Fragment 2 from mTlaA1) were analyzed using 1D/2D NMR (TOCSY, NOESY, HSQC).
• Stereochemistry Determination: Marfey's analysis (acid hydrolysis followed by L-FDLA derivatization) was performed to determine the stereochemistry (LL vs. DL) of the lanthionine (Lan) and methyllanthionine (MeLan) rings.
• Bioactivity Assays: Agar well-diffusion assays were conducted against Bacillus subtilis and ESKAPE pathogens (including Gram-negatives) using native cleavage products and AspN-digested fragments.

3. Key Results

A. Structural Modification and Ring Patterns

• Dehydration: TlaM dehydrated TlaA1 seven times and TlaA2 six times.
• Cyclization: Both peptides formed four thioether rings.
  • mTlaA1: Contains rings A, B, C, and D.
  • mTlaA2: Contains rings A, B, C, and D.
• Unique Topology: The C-terminus features a sequence (SSXATPCKRC) where two of the three Ser/Thr residues are dehydrated, leading to two overlapping rings at the C-terminus. NMR confirmed the specific connectivity of these overlapping rings (Rings B, C, and D).
• Congeners: The two peptides are structural congeners (very similar structures) rather than distinct partners required for synergy.

B. Stereochemical Anomaly (Key Finding)

• Deviation from Rule: Standard class II synthetases produce LL-(methyl)lanthionine from the DhxDhxXxxXxxCys motif.
• Discovery: Marfey's analysis revealed that TlaM produces DL-(methyl)lanthionine rings.
  • Both mTlaA1 and mTlaA2 contain DL-Lan and DL-MeLan.
  • Specifically, the N-terminal "A-ring" (formed from a Dhb-Dha-Leu-Val-Cys motif) was confirmed to be DL-MeLan.
• Significance: This is a rare case where the enzyme enforces a stereochemical outcome opposite to the inherent preference of the substrate motif, suggesting a unique catalytic mechanism for TlaM.

C. Bioactivity

• Native Products: The leader-peptide-cleaved mTlaA1 and mTlaA2 showed weak to no activity individually or in combination.
• Second Proteolysis Hypothesis: Similar to other two-component lantibiotics, a second proteolytic step (removing 6–7 N-terminal residues) is likely required for full activity.
• Active Fragment: AspN digestion of mTlaA1 (removing the N-terminal 6 residues) yielded a 31-mer peptide (named Thermolanthin A) that displayed potent antimicrobial activity against B. subtilis and, notably, Gram-negative ESKAPE pathogens (Acinetobacter baumanii, Klebsiella pneumoniae).
• Implication: The two peptides likely function as a single-component system (congeners) rather than a synergistic two-component system, as the active fragment of mTlaA1 functions alone.

4. Contributions

1. Discovery of Thermolanthin: Identification and structural characterization of a new class II lanthipeptide from a thermophilic bacterium.
2. Structural Novelty: Elucidation of a unique ring topology involving overlapping rings at the C-terminus, a pattern not previously reported in known lanthipeptides.
3. Stereochemical Breakthrough: Demonstration that a class II synthetase (TlaM) can catalyze the formation of DL-(methyl)lanthionine rings, challenging the established dogma that DhxDhx motifs exclusively yield LL-rings.
4. Bioactivity Insight: Identification of a Gram-negative active lantibiotic derivative (Thermolanthin A), highlighting the potential of thermophilic RiPPs as sources for new antibiotics against multidrug-resistant pathogens.

5. Significance

This study expands the known structural and stereochemical diversity of lanthipeptides. The discovery of DL-stereochemistry enforced by TlaM suggests that the "rules" of lanthipeptide biosynthesis are more flexible than previously thought, opening new avenues for bioengineering enzymes to produce non-natural stereochemical variants. Furthermore, the ability of Thermolanthin A to inhibit Gram-negative ESKAPE pathogens addresses a critical need in antibiotic development, as lanthipeptides typically struggle to penetrate the outer membrane of Gram-negative bacteria. The findings also suggest that the tla BGC produces congeners rather than a strict two-component system, refining the understanding of how multi-precursor BGCs evolve and function.